Exploring the performance of multi-channel Tetrahedral Nucleic Acid Tweezers platforms for efficient and sensitive biosensing

• Molecular dynamics simulation of the CTNAT and the ATNAT reporter. • Multi-channel detection and background signal of the ATNAT and the CTNAT reporter. • Redesigns of the CTNAT reporter in the nanosize and the hybridization efficiency. • EXPAR-cDNA-CTNAT strategy for the detection of three fatigue biomarkers. • Accurate, sensitive and specific fatigue diagnosis. The nucleic tweezers-based detection strategies have shown excellent application prospects in molecular detection and diagnosis thanks to the specific target responsiveness and good biocompatibility. However, the limited detection efficiency and unsatisfactory sensitivity make their applications in complex diagnosis difficult, and few reports focused on it. Here, we explored the molecular dynamics simulations and biosensing performance of the Tetrahedral Nucleic Acid Tweezers, including the Antennae like-Tetrahedron Nucleic Acid Tweezer (ATNAT) and the Covered-Tetrahedron Nucleic Acid Tweezer (CTNAT), revealing the molecular dynamics of ATNAT and CTNAT reporters visually and molecularly. After that, we compared the multi-target detection capabilities of DNA tetrahedral tweezers, and combined the CTNAT reporter with better multi-target detection performance with the aptamer and Exponential amplification reaction (EXPAR), developing an efficient and sensitive EXPAR-cDNA-CTNAT strategy. Then we applied the EXPAR-cDNA-CTNAT strategy to detect testosterone, cortisol, and creatine kinase isoenzymes, realizing sensitive and accurate fatigue diagnosis. Compared with the traditional detection strategies, the EXPAR-cDNA-CTNAT strategy showed improved sensitivity and detection efficiency with excellent specificity, and the limits of detection (LODs) for the multi-target detection were as low as 41, 68, and 8 pM, respectively. The EXPAR-cDNA-CTNAT strategy was reliable for multi-target detection, which had great potential in biological science, food safety, and medical diagnosis.